Transferrin/transferrin receptor-mediated siRNA delivery

ABSTRACT

The invention provides interfering RNA molecule-ligand conjugates useful as a delivery system for delivering interfering RNA molecules to a cell in vitro or in vivo. The conjugates comprise a ligand that can bind to a transferrin receptor (TfR). Therapeutic uses for the conjugates are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 13/112,635 filed May 20, 2011 (now allowed), which claims priorityto U.S. patent application Ser. No. 12/244,027 filed Oct. 2, 2008 (nowU.S. Pat. No. 7,973,019), which claims benefit to Provisional PatentApplication No. 60/977,272 filed Oct. 3, 2007, the entire contents ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods of delivering interfering RNA moleculesto a cell via interfering RNA molecule-ligand conjugates. The conjugatescomprise a ligand that can bind to a transferrin receptor (TfR). Theinvention also relates to methods for treating ocular disorders byadministering an interfering RNA molecule-ligand conjugate of theinvention to a patient in need thereof.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a process by which double-stranded RNA(dsRNA) is used to silence gene expression. RNAi is induced by short(i.e. <30 nucleotide) double stranded RNA (“dsRNA”) molecules which arepresent in the cell (Fire et al., 1998, Nature 391:806-811). These shortdsRNA molecules called “short interfering RNA” or “siRNA,” cause thedestruction of messenger RNAs (“mRNAs”) which share sequence homologywith the siRNA (Elbashir et al., 2001, Genes Dev, 15:188-200). It isbelieved that one strand of the siRNA is incorporated into aribonucleoprotein complex known as the RNA-induced silencing complex(RISC). RISC uses this siRNA strand to identify mRNA molecules that areat least partially complementary to the incorporated siRNA strand, andthen cleaves these target mRNAs or inhibits their translation. The siRNAis apparently recycled much like a multiple-turnover enzyme, with 1siRNA molecule capable of inducing cleavage of approximately 1000 mRNAmolecules. siRNA-mediated RNAi degradation of an mRNA is therefore moreeffective than currently available technologies for inhibitingexpression of a target gene.

RNAi provides a very exciting approach to treating and/or preventingdiseases. Some major benefits of RNAi compared with various traditionaltherapeutic approaches include: the ability of RNAi to target a veryparticular gene involved in the disease process with high specificity,thereby reducing or eliminating off target effects; RNAi is a normalcellular process leading to a highly specific RNA degradation; and RNAidoes not trigger a host immune response as in many antibody basedtherapies.

Several interfering RNA delivery methods are being tested/developed forin vivo use. For example, siRNAs can be delivered “naked” in salinesolution; complexed with polycations, cationic lipids/lipid transfectionreagents, or cationic peptides; as components of defined molecularconjugates (e.g., cholesterol-modified siRNA, TAT-DRBD/siRNA complexes);as components of liposomes; and as components of nanoparticles. Theseapproaches have shown varying degrees of success. Thus, there is a needfor new and improved methods for delivering siRNA molecules in vivo toachieve and enhance the therapeutic potential of RNAi.

SUMMARY OF THE INVENTION

The invention provides interfering RNA molecule-ligand conjugates,wherein the ligand can bind to a transferrin receptor (TfR). Theinvention also provides methods of using the conjugates for deliveringan interfering RNA molecule into a cell in vitro or in vivo. In oneaspect, an interfering RNA molecule-ligand conjugate of the inventioncan be used deliver an interfering RNA molecule to an eye of a patient.

The invention further provides methods of treating or preventing anocular disorder in a patient, comprising administering to the patient aninterfering RNA molecule-ligand conjugate, wherein the ligand can bindto a transferrin receptor (TfR) and wherein the interfering RNA moleculecan attenuate expression of a gene associated with the ocular disorder.In certain aspects, the ocular disorder is associated with ocularangiogenesis, dry eye, ocular inflammatory conditions, ocularhypertension, or glaucoma. In other aspects, the conjugate isadministered by intraocular injection, subconjunctival injection,intravitreal injection, anterior or posterior juxtascleral injection,topical ocular application, intravenous injection, oral administration,intramuscular injection, intraperitoneal injection, transdermalapplication, intranasal application, or transmucosal application.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

DETAILED DESCRIPTION OF THE INVENTION

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

The following definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3^(rd) Edition or a dictionary known to those of skill inthe art, such as the Oxford Dictionary of Biochemistry and MolecularBiology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

As used herein, all percentages are percentages by weight, unless statedotherwise.

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one”, “at least one” or “one or more”. Unlessotherwise required by context, singular terms used herein shall includepluralities and plural terms shall include the singular.

In certain embodiments, the invention provides interfering RNA-ligandconjugates that can deliver interfering RNAs into an eye cell of apatient. In a particular embodiment, the conjugates can bind to atransferrin receptor (TfR) on the surface of an eye cell. Thetransferrin receptor is an integral membrane glycoprotein that mediatesthe uptake of iron by individual cells. There appears to be acorrelation between the number of receptors on the surface of a cell andcellular proliferation, with the highest number of receptors being onactively growing cells and the lowest number being on resting andterminally differentiated cells.

The term “receptor” as used herein is intended to encompass the entirereceptor or ligand-binding portions thereof. These portions of thereceptor particularly include those regions sufficient for specificbinding of the ligand to occur.

The ligand of the conjugate can be any molecule that is capable ofbinding with specificity to the transferrin receptor on cells of theeye. Examples of molecules include, but are not limited to, proteins andaptamers.

The term “protein” as used herein includes peptides, polypeptides,consensus molecules, fusion proteins, purified naturally occurringproteins, artificially synthesized proteins, antibodies, and analogs,derivatives or combinations thereof.

The term “aptamer” as used herein refers to nucleic acids (typicallyDNA, RNA or oligonucleotides) that are capable of binding to aparticular molecular target. Aptamers emerge from in vitro selections orother types of aptamer selection procedures well known in the art (e.g.bead-based selection with flow cytometry or high density aptamer arrays)when the nucleic acid is added to mixtures of target molecules. Anaptamer is typically between 10 and 300 nucleotides in length. RNA andDNA aptamers can be generated from in vitro selection experiments suchas SELEX (Systematic Evolution of Ligands by Exponential Enrichment).Examples of aptamer uses and methods for making/selecting aptamers aredescribed, for example, in Chu et al., 2006, Nucl. Acids Res. 34:e73),U.S. Patent Publication No. 20060014172, U.S. Pat. Nos. 5,840,867,6,001,648, 6225,058, 6,207,388, and U.S. Patent Publication No.20020001810, the disclosures of all of which are incorporated byreference in their entireties.

A particularly preferred ligand family includes peptides comprising theTfR-binding domain of transferrin (Tf), which is the nominal TfR ligand.In certain embodiments, the ligand is full length Tf, a less than fulllength Tf protein that comprises a TfR binding domain, or the C-lobe ofTf or a TfR-binding portion thereof.

Monoclonal antibody binding (Teh et al. FEBS J. 272:6344-6353, 2005) andhydroxyl radical-mediated protein footprinting (Liu et al. Biochemistry42:12447-12454, 2003) suggest that the TfR-binding domain of Tf islocated within the C-lobe, residues 334-679 of human Tf. Residues365-401 (especially 381-401), 415-433, and 457-470 appear to beparticularly important for receptor binding.

An interfering RNA molecule can be covalently linked to a TfR-bindingdomain either directly or via a spacer, such as a glycine spacer of 1,2, 3, or 4 glycines. Preferably, a glycine spacer is 2 or 3 glycines.

Other examples of ligands that can bind with specificity to TfR includeantibodies or antibody fragments that can bind TfR. These antibodies orantibody fragments are as capable of binding to TfR as the nominalreceptor ligand. Upon binding of the antibodies to TfR on a cellsurface, transfer of the antibody and the attached interfering RNA intothe cell occurs. The interfering RNA can be attached by any acceptablemeans for joining the antibody to the interfering RNA such that theinterfering RNA can be transferred across the cell membrane in apharmaceutically active form. In a preferred embodiment, theTfR-specific antibody or antibody fragment forms a conjugate with theinterfering RNA.

In other embodiments, a TfR-specific antibody or antibody fragment and asecond ligand, which is also reactive with the TfR, are joined togetherto form a fusion protein. The second ligand can be a second antibody or,more preferably, a nominal ligand such as transferrin, or a TfR bindingfragment thereof. These fusion proteins have the advantage of possessingthe capacity of interacting twice as readily with ocular cells thanconjugates that only have one ligand.

Antibodies that can be used in this invention are reactive with atransferrin receptor on an eye cell. The term antibody is intended toencompass both polyclonal and monoclonal antibodies. The term antibodyis also intended to encompass mixtures of more than one antibodyreactive with a transferrin receptor (e.g., a cocktail of differenttypes of monoclonal antibodies reactive with the TfR), each of which isjoined to an interfering RNA to form a conjugate. The term antibody isfurther intended to encompass whole antibodies, biologically functionalfragments thereof, fully humanized antibodies, and chimeric antibodiescomprising portions from more than one species, bifunctional antibodies,etc. Biologically functional antibody fragments which can be used arethose fragments which can be used for binding of the antibody fragmentto the TfR to occur. Examples of TfR antibodies are described, forexample, in Callens et al. (Leukemia, 27 Sep. 2007 epub ahead of print)and Qian et al. (Pharmacol. Rev. 54:561-587, 2002).

The interfering RNA can be linked to a ligand using chemical conjugationtechniques. In addition to covalent bonding, conjugates can be formedemploying non-covalent bonds, such as those formed with bifunctionalantibodies, ionic bonds, hydrogen bonds, hydrophobic interactions, etc.

In certain embodiments, an interfering RNA-ligand conjugate of theinvention can further comprise a nucleic acid binding protein, such asprotamine, covalently linked to the ligand. For example, the ligand ofthe conjugate can comprise a transferrin peptide-protamine fusionprotein or a TfR-specific antibody-protamine fusion protein.Antibody-protamine fusion proteins have been used to deliver siRNA toHIV-infected or envelope-transfected cells (Song et al., 2005, Nat.Biotechnol. 23:709-717). The interfering RNA molecule can be linked tothe ligand via interaction with the nucleic acid binding protein.

In other embodiments, the ligand of the interfering RNA-ligand conjugateof the invention is covalently linked to a polycation, such aspolylysine. For example, the conjugate can comprise a transferrinpeptide fused to polylysine or another polycation, or a TfR-specificantibody fused to polylysine or another polycation, such as polyarginineor polyethyleneimine (PEI). Methods for preparing and delivering nucleicacids to a variety of cultured mammalian cells and to tumor-bearing miceusing transferrin-polycation-DNA conjugates have been described(reviewed in Qian, et al., 2002, Pharmacol Rev 54:561-587). Theinterfering RNA molecule can be linked to the ligand via interactionwith the polycation.

In certain embodiments, the interfering RNA molecule is linked to theligand via a peptide consisting entirely of arginines (referred toherein as an “Arg peptide”). Preferably, the Arg peptide comprises 7arginines (7×Arg), 8 arginines (8×Arg), 9 arginines (9×Arg), 10arginines (10×Arg), or 11 (11×Arg) arginines. The Arg peptide can belinked to the C- or N-terminus of a ligand, such as the TfR-bindingdomain, via a glycine spacer of 1 to 4 glycines. Preferably, the glycinespacer is 2 or 3 glycines.

In one embodiment, the Arg peptide is a 9×Arg peptide. In oneembodiment, the 9×Arg peptide comprises or consists of D-isomers. In aparticular embodiment, the “9×Arg peptide” as used herein means apeptide of 9 arginine residues(R^(†)R^(\)R^(\)R^(\)R^(\)R^(\)R^(\)R^(\)R^(\); SEQ ID NO: 1).Negatively charged interfering RNA molecules can bind to the positivelycharged 9×Arg peptide as described in Kumar et al., who recentlydemonstrated that a 9×Arg peptide could be used to link interfering RNAmolecules to the C-terminal end of a rabies virus glycoprotein (RVG)targeting peptide for delivery across the blood-brain barrier (Kumar etal., Jun. 17, 2007, Nature, epub ahead of print).

In certain embodiments, an interfering RNA-ligand conjugate isadministered to a patient or a cell in the presence of a TAT-HA2peptide, a ligand-HA2 peptide, or a retro-inverso (i.e., the reversesequence constructed of D-amino acids) TAT-HA2 peptide, which has beenshown to enhance release of peptide/protein conjugates from the endosome(Wadia et al., 2004, Nat. Med. 10:310). The term “HA2 peptide” means apeptide comprising the N-terminal 20 amino acids of influenza virushemagglutinin protein. The native HA2 peptide is:

SEQ ID NO: 2 GLFGAIAGFIENGWEGMIDG;.

Preferably, the native HA2 peptide comprises L-isomers.

The retro-inverso HA2 peptide is:

SEQ ID NO: 3GD^(†)I^(†)M^(†)GE^(†)W^(†)GN^(†)E^(†)I^(†)F^(†)GA^(†)I^(†)A^(†)GF^(†)L^(†)G;.

D-isomers are denoted by a superscripted dagger (^(\)) to the right ofthe one-letter code symbol; thus, D^(\) represents D-aspartic acid andL^(\) represents D-leucine.

The presence of HA2 aids release of the interfering RNA delivery systemfrom the endosome into the cytosol, so that the interfering RNAmolecular can attenuate expression of a target mRNA in a cell. Incertain other embodiments, an HA2 peptide is inserted between the TfRligand and the 9×Arg, wherein the HA2 peptide is linked to the ligandand 9×Arg via glycine spacers. One example of a ligand-HA2 conjugate isas follows:

SEQ ID NO: 4Ligand-GGGD^(†)I^(†)M^(†)GE^(†)W^(†)GN^(†)E^(†)I^(†)F^(†)GA^(†)I^(†)A^(†)GF^(†)L^(†)GGGR^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†)R^(†);.

In certain embodiments, the TfR ligand-9×Arg peptide is produced as asingle peptide before being conjugated to the interfering RNA molecule.In other embodiments, the TfR ligand-9×Arg peptide can be produced bycombining the TfR ligand and the 9×Arg peptide under conditions in whichthe ligand and the peptide will connect to each other. Such methods forlinking two peptides are well known in the art. In yet otherembodiments, the 9×Arg peptide can be premixed with the interfering RNAmolecule and then linked to the TfR ligand to favor binding of theinterfering RNA to the 9×Arg end of the peptide. Thus, linkage of theinterfering RNA molecule can be accomplished before or after linkage ofTfR ligand with 9×Arg.

In certain embodiments, the invention provides a method of attenuatingexpression of a target mRNA in an eye of a patient, comprising (a)providing an interfering RNA-ligand conjugate, wherein the conjugatebinds to a transferrin receptor (TfR); and (b) administering theconjugate to an eye of the patient, wherein the interfering RNA moleculecan attenuate expression of the target mRNA in the eye.

In certain embodiments, the invention provides a method of preventing ortreating an ocular disorder in a patient, the method comprisingadministering to the patient an interfering RNA-ligand conjugate,wherein the conjugate binds to a TfR and transports said interfering RNAinto an eye cell of the patient.

The term “patient” as used herein means a human or other mammal havingan ocular disorder or at risk of having an ocular disorder. Ocularstructures associated with such disorders may include the eye, retina,choroid, lens, cornea, trabecular meshwork (TM), iris, optic nerve,optic nerve head, sclera, anterior or posterior segment, or ciliarybody, for example. In certain embodiments, a patient has an oculardisorder associated with TM cells, ciliary epithelium cells, or anothercell type in the eye.

The term “ocular disorder” as used herein includes conditions associatedwith ocular angiogenesis, dry eye, inflammatory conditions, ocularhypertension and ocular diseases associated with elevated intraocularpressure (TOP), such as glaucoma.

The term “ocular angiogenesis,” as used herein, includes ocularpre-angiogenic conditions and ocular angiogenic conditions, and includesthose cellular changes resulting from the expression of certain genesthat lead directly or indirectly to ocular angiogenesis, ocularneovascularization, retinal edema, diabetic retinopathy, sequelaassociated with retinal ischemia, posterior segment neovascularization(PSNV), and neovascular glaucoma, for example. The interfering RNAs usedin a method of the invention are useful for treating patients withocular angiogenesis, ocular neovascularization, retinal edema, diabeticretinopathy, sequela associated with retinal ischemia, posterior segmentneovascularization (PSNV), and neovascular glaucoma, or patients at riskof developing such conditions, for example. The term “ocularneovascularization” includes age-related macular degeneration, cataract,acute ischemic optic neuropathy (AION), commotio retinae, retinaldetachment, retinal tears or holes, iatrogenic retinopathy and otherischemic retinopathies or optic neuropathies, myopia, retinitispigmentosa, and/or the like.

The term “inflammatory condition,” as used herein, includes conditionssuch as ocular inflammation, allergic conjunctivitis, dermatitis,rhinitis, and asthma.

The methods of the invention are useful for attenuating expression ofparticular genes in the eyes of patients using RNA interference.

RNA interference (RNAi) is a process by which double-stranded RNA(dsRNA) is used to silence gene expression. While not wanting to bebound by theory, RNAi begins with the cleavage of longer dsRNAs intosmall interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer.SiRNAs are dsRNAs that are usually about 19 to 28 nucleotides, or 20 to25 nucleotides, or 21 to 22 nucleotides in length and often contain2-nucleotide 3′ overhangs, and 5′ phosphate and 3′ hydroxyl termini. Onestrand of the siRNA is incorporated into a ribonucleoprotein complexknown as the RNA-induced silencing complex (RISC). RISC uses this siRNAstrand to identify mRNA molecules that are at least partiallycomplementary to the incorporated siRNA strand, and then cleaves thesetarget mRNAs or inhibits their translation. Therefore, the siRNA strandthat is incorporated into RISC is known as the guide strand or theantisense strand. The other siRNA strand, known as the passenger strandor the sense strand, is eliminated from the siRNA and is at leastpartially homologous to the target mRNA. Those of skill in the art willrecognize that, in principle, either strand of an siRNA can beincorporated into RISC and function as a guide strand. However, siRNAdesign (e.g., decreased siRNA duplex stability at the 5′ end of thedesired guide strand) can favor incorporation of the desired guidestrand into RISC.

The antisense strand of an siRNA is the active guiding agent of thesiRNA in that the antisense strand is incorporated into RISC, thusallowing RISC to identify target mRNAs with at least partialcomplementarity to the antisense siRNA strand for cleavage ortranslational repression. RISC-mediated cleavage of mRNAs having asequence at least partially complementary to the guide strand leads to adecrease in the steady state level of that mRNA and of the correspondingprotein encoded by this mRNA. Alternatively, RISC can also decreaseexpression of the corresponding protein via translational repressionwithout cleavage of the target mRNA.

Interfering RNAs appear to act in a catalytic manner for cleavage oftarget mRNA, i.e., interfering RNA is able to effect inhibition oftarget mRNA in substoichiometric amounts. As compared to antisensetherapies, significantly less interfering RNA is required to provide atherapeutic effect under such cleavage conditions.

In certain embodiments, the invention provides methods of deliveringinterfering RNA to inhibit the expression of a target mRNA thusdecreasing target mRNA levels in patients with ocular disorders.

The phrase “attenuating expression” with reference to a gene or an mRNAas used herein means administering or expressing an amount ofinterfering RNA (e.g., an siRNA) to reduce translation of a target mRNAinto protein, either through mRNA cleavage or through direct inhibitionof translation. The terms “inhibit,” “silencing,” and “attenuating” asused herein refer to a measurable reduction in expression of a targetmRNA or the corresponding protein as compared with the expression of thetarget mRNA or the corresponding protein in the absence of aninterfering RNA of the invention. The reduction in expression of thetarget mRNA or the corresponding protein is commonly referred to as“knock-down” and is reported relative to levels present followingadministration or expression of a non-targeting control RNA (e.g., anon-targeting control siRNA). Knock-down of expression of an amountincluding and between 50% and 100% is contemplated by embodimentsherein. However, it is not necessary that such knock-down levels beachieved for purposes of the present invention.

Knock-down is commonly assessed by measuring the mRNA levels usingquantitative polymerase chain reaction (qPCR) amplification or bymeasuring protein levels by western blot or enzyme-linked immunosorbentassay (ELISA). Analyzing the protein level provides an assessment ofboth mRNA cleavage as well as translation inhibition. Further techniquesfor measuring knock-down include RNA solution hybridization, nucleaseprotection, northern hybridization, gene expression monitoring with amicroarray, antibody binding, radioimmunoassay, and fluorescenceactivated cell analysis.

Attenuating expression of a target gene by an interfering RNA moleculeof the invention can be inferred in a human or other mammal by observingan improvement in symptoms of the ocular disorder, including, forexample, a decrease in intraocular pressure that would indicateinhibition of a glaucoma target gene.

In one embodiment, a single interfering RNA is delivered to decreasetarget mRNA levels. In other embodiments, two or more interfering RNAstargeting the mRNA are administered to decrease target mRNA levels. Theinterfering RNAs may be delivered in the same interfering RNAmolecule-ligand conjugate or in separate conjugates.

As used herein, the terms “interfering RNA” and “interfering RNAmolecule” refer to all RNA or RNA-like molecules that can interact withRISC and participate in RISC-mediated changes in gene expression.Examples of other interfering RNA molecules that can interact with RISCinclude siRNAs, short hairpin RNAs (shRNAs), single-stranded siRNAs,microRNAs (miRNAs), and dicer-substrate 27-mer duplexes. Examples of“RNA-like” molecules that can interact with RISC include siRNA,single-stranded siRNA, microRNA, and shRNA molecules that contain one ormore chemically modified nucleotides, one or more non-nucleotides, oneor more deoxyribonucleotides, and/or one or more non-phosphodiesterlinkages. Thus, siRNAs, single-stranded siRNAs, shRNAs, miRNAs, anddicer-substrate 27-mer duplexes are subsets of “interfering RNAs” or“interfering RNA molecules.”

The term “siRNA” as used herein refers to a double-stranded interferingRNA unless otherwise noted. Typically, an siRNA used in a method of theinvention is a double-stranded nucleic acid molecule comprising twonucleotide strands, each strand having about 19 to about 28 nucleotides(i.e. about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides).Typically, an interfering RNA used in a method of the invention has alength of about 19 to 49 nucleotides. The phrase “length of 19 to 49nucleotides” when referring to a double-stranded interfering RNA meansthat the antisense and sense strands independently have a length ofabout 19 to about 49 nucleotides, including interfering RNA moleculeswhere the sense and antisense strands are connected by a linkermolecule.

Single-stranded interfering RNA has been found to effect mRNA silencing,albeit less efficiently than double-stranded RNA. Therefore, embodimentsof the present invention also provide for administration of asingle-stranded interfering RNA. The single-stranded interfering RNA hasa length of about 19 to about 49 nucleotides as for the double-strandedinterfering RNA cited above. The single-stranded interfering RNA has a5′ phosphate or is phosphorylated in situ or in vivo at the 5′ position.The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the sugar (e.g., ribose,deoxyribose, or an analog of same) at the 5′ end of the polynucleotideor oligonucleotide.

Single-stranded interfering RNAs can be synthesized chemically or by invitro transcription or expressed endogenously from vectors or expressioncassettes as described herein in reference to double-strandedinterfering RNAs. 5′ Phosphate groups may be added via a kinase, or a 5′phosphate may be the result of nuclease cleavage of an RNA. A hairpininterfering RNA is a single molecule (e.g., a single oligonucleotidechain) that comprises both the sense and antisense strands of aninterfering RNA in a stem-loop or hairpin structure (e.g., a shRNA). Forexample, shRNAs can be expressed from DNA vectors in which the DNAoligonucleotides encoding a sense interfering RNA strand are linked tothe DNA oligonucleotides encoding the reverse complementary antisenseinterfering RNA strand by a short spacer. If needed for the chosenexpression vector, 3′ terminal T's and nucleotides forming restrictionsites may be added. The resulting RNA transcript folds back onto itselfto form a stem-loop structure.

Interfering RNAs may differ from naturally-occurring RNA by theaddition, deletion, substitution or modification of one or morenucleotides. Non-nucleotide material may be bound to the interferingRNA, either at the 5′ end, the 3′ end, or internally. Such modificationsare commonly designed to increase the nuclease resistance of theinterfering RNAs, to improve cellular uptake, to enhance cellulartargeting, to assist in tracing the interfering RNA, to further improvestability, or to reduce the potential for activation of the interferonpathway. For example, interfering RNAs may comprise a purine nucleotideat the ends of overhangs. Conjugation of cholesterol to the 3′ end ofthe sense strand of an siRNA molecule by means of a pyrrolidine linker,for example, also provides stability to an siRNA.

Further modifications include a 5′ or 3′ terminal biotin molecule, apeptide known to have cell-penetrating properties, a nanoparticle, apeptidomimetic, a fluorescent dye, or a dendrimer, for example.

Nucleotides may be modified on their base portion, on their sugarportion, or on the phosphate portion of the molecule and function inembodiments of the present invention. Modifications includesubstitutions with alkyl, alkoxy, amino, deaza, halo, hydroxyl, thiolgroups, or a combination thereof, for example. Nucleotides may besubstituted with analogs with greater stability such as replacing aribonucleotide with a deoxyribonucleotide, or having sugar modificationssuch as 2′ OH groups replaced by 2′ amino groups, 2′ O-methyl groups, 2′methoxyethyl groups, or a 2′-O, 4′-C methylene bridge, for example.Examples of a purine or pyrimidine analog of nucleotides include axanthine, a hypoxanthine, an azapurine, a methylthioadenine,7-deaza-adenosine and O- and N-modified nucleotides. The phosphate groupof the nucleotide may be modified by substituting one or more of theoxygens of the phosphate group with nitrogen or with sulfur(phosphorothioates). Modifications are useful, for example, to enhancefunction, to improve stability or permeability, or to directlocalization or targeting.

In certain embodiments, an interfering molecule of the inventioncomprises at least one of the modifications as described above.

The phrases “target sequence” and “target mRNA” as used herein refer tothe mRNA or the portion of the mRNA sequence that can be recognized byan interfering RNA used in a method of the invention, whereby theinterfering RNA can silence gene expression as discussed herein.Techniques for selecting target sequences for siRNAs are provided, forexample, by Tuschl, T. et al., “The siRNA User Guide,” revised May 6,2004, available on the Rockefeller University web site; by TechnicalBulletin #506, “siRNA Design Guidelines,” Ambion Inc. at Ambion's website; and by other web-based design tools at, for example, theInvitrogen, Dharmacon, Integrated DNA Technologies, Genscript, orProligo web sites. Initial search parameters can include G/C contentsbetween 35% and 55% and siRNA lengths between 19 and 27 nucleotides. Thetarget sequence may be located in the coding region or in the 5′ or 3′untranslated regions of the mRNA. The target sequences can be used toderive interfering RNA molecules, such as those described herein.

Interfering RNA target sequences (e.g., siRNA target sequences) within atarget mRNA sequence are selected using available design tools asdescribed above. Interfering RNAs corresponding to a target sequence arethen tested in vitro by transfection of cells expressing the target mRNAfollowed by assessment of knockdown as described herein. The interferingRNAs can be further evaluated in vivo using animal models as describedherein.

The ability of interfering RNA to knock-down the levels of endogenoustarget gene expression in, for example, HeLa cells can be evaluated invitro as follows. HeLa cells are plated 24 h prior to transfection instandard growth medium (e.g., DMEM supplemented with 10% fetal bovineserum). Transfection is performed using, for example, Dharmafect 1(Dharmacon, Lafayette, Colo.) according to the manufacturer'sinstructions at interfering RNA concentrations ranging from 0.1 nM-100nM. SiCONTROL™ Non-Targeting siRNA #1 and siCONTROL™ Cyclophilin B siRNA(Dharmacon) are used as negative and positive controls, respectively.Target mRNA levels and cyclophilin B mRNA (PPIB, NM_(—)000942) levelsare assessed by qPCR 24 h post-transfection using, for example, aTAQMAN® Gene Expression Assay that preferably overlaps the target site(Applied Biosystems, Foster City, Calif.). The positive control siRNAgives essentially complete knockdown of cyclophilin B mRNA whentransfection efficiency is 100%. Therefore, target mRNA knockdown iscorrected for transfection efficiency by reference to the cyclophilin BmRNA level in cells transfected with the cyclophilin B siRNA. Targetprotein levels may be assessed approximately 72 h post-transfection(actual time dependent on protein turnover rate) by western blot, forexample. Standard techniques for RNA and/or protein isolation fromcultured cells are well-known to those skilled in the art. To reduce thechance of non-specific, off-target effects, the lowest possibleconcentration of interfering RNA is used that produces the desired levelof knock-down in target gene expression. Human corneal epithelial cells,trabecular meshwork cells, ciliary epithelial cells, retinal pigmentepithelial cells, or other human ocular cell lines may also be use foran evaluation of the ability of interfering RNA to knock-down levels ofan endogenous target gene.

A number of animal models are known that can be used to test theactivity of an interfering RNA molecule. For example, siRNA moleculescan be tested in murine laser-induced models of choroidalneovascularization (CNV) as described in Reich et al., 2003, Mol.Vision. 9:210-216; Shen et al., 2006, Gene Therapy 13:225-234; or Boraet al., 2006, J. Immunol. 177:1872-1878.

In certain embodiments, an interfering RNA molecule-ligand conjugatecomprises an interfering RNA molecule that targets a gene associatedwith an ocular disorder. Examples of mRNA target genes for whichinterfering RNAs of the present invention are designed to target includegenes associated with the disorders that affect the retina, genesassociated with glaucoma, and genes associated with ocular inflammation.

Examples of mRNA target genes associated with the retinal disordersinclude tyrosine kinase, endothelial (TEK); complement factor B (CFB);hypoxia-inducible factor 1, α subunit (HIF1A); HtrA serine peptidase 1(HTRA1); platelet-derived growth factor receptor β (PDGFRB); chemokine,CXC motif, receptor 4 (CXCR4); insulin-like growth factor I receptor(IGF1R); angiopoietin 2 (ANGPT2); v-fos FBJ murine osteosarcoma viraloncogene homolog (FOS); cathepsin L1, transcript variant 1 (CTSL1);cathepsin L1, transcript variant 2 (CTSL2); intracellular adhesionmolecule 1 (ICAM1); insulin-like growth factor I (IGF1); integrin α5(ITGAS); integrin β1 (ITGB1); nuclear factor kappa-B, subunit 1 (NFKB1);nuclear factor kappa-B, subunit 2 (NFKB2); chemokine, CXC motif, ligand12 (CXCL12); tumor necrosis factor-alpha-converting enzyme (TACE); tumornecrosis factor receptor 1 (TNFR1); vascular endothelial growth factor(VEGF); vascular endothelial growth factor receptor 1 (VEGFR1); andkinase insert domain receptor (KDR).

Examples of target genes associated with glaucoma include carbonicanhydrase II (CA2); carbonic anhydrase IV (CA4); carbonic anhydrase XII(CA12); β1 andrenergic receptor (ADBR1); β2 andrenergic receptor(ADBR2); acetylcholinesterase (ACHE); Na+/K+− ATPase; solute carrierfamily 12 (sodium/potassium/chloride transporters), member 1 (SLC12A1);solute carrier family 12 (sodium/potassium/chloride transporters),member 2 (SLC12A2); connective tissue growth factor (CTGF); serumamyloid A (SAA); secreted frizzled-related protein 1 (sFRP1); gremlin(GREM1); lysyl oxidase (LOX); c-Maf; rho-associatedcoiled-coil-containing protein kinase 1 (ROCK1); rho-associatedcoiled-coil-containing protein kinase 2 (ROCK2); plasminogen activatorinhibitor 1 (PAI-1); endothelial differentiation, sphingolipidG-protein-coupled receptor, 3 (Edg3 R); myocilin (MYOC); NADPH oxidase 4(NOX4); Protein Kinase Cδ (PKCδ); Aquaporin 1 (AQP1); Aquaporin 4(AQP4); members of the complement cascade; ATPase, H+ transporting,lysosomal V1 subunit A (ATP6V1A); gap junction protein α-1 (GJA1);formyl peptide receptor 1 (FPR1); formyl peptide receptor-like 1(FPRL1); interleukin 8 (IL8); nuclear factor kappa-B, subunit 1 (NFKB1);nuclear factor kappa-B, subunit 2 (NFKB2); presenilin 1 (PSEN1); tumornecrosis factor-alpha-converting enzyme (TACE); transforming growthfactor β2 (TGFB2); transient receptor potential cation channel,subfamily V, member 1 (TRPV1); chloride channel 3 (CLCN3); gap junctionprotein α5 (GJAS); tumor necrosis factor receptor 1 (TNFR1); andchitinase 3-like 2 (CHI3L2).

Examples of mRNA target genes associated with ocular inflammationinclude tumor necrosis factor receptor superfamily, member 1A(TNFRSF1A); phosphodiesterase 4D, cAMP-specific (PDE4D); histaminereceptor H1 (HRH1); spleen tyrosine kinase (SYK); interkeukin 1β (IL1B);nuclear factor kappa-B, subunit 1 (NFKB1); nuclear factor kappa-B,subunit 2 (NFKB2); and tumor necrosis factor-alpha-converting enzyme(TACE).

Such target genes are described, for example, in U.S. PatentApplications having Publication Nos. 20060166919, 20060172961,20060172963, 20060172965, 20060223773, 20070149473, and 20070155690, thedisclosures of which are incorporated by reference in their entirety.

In other embodiments, the method of delivering an interfering RNAmolecule comprises administering to the patient a nanoparticle-ligandconjugate, wherein the interfering RNA molecule is encapsulated in thenanoparticle and the nanoparticle is linked to a ligand that can bind tothe transferrin receptor, which transports the interfering RNA moleculeinto an eye cell of the patient. Other embodiments of the inventionprovide a method of preventing or treating an ocular disorder, saidmethod comprising delivering an interfering RNA molecule to the eye of apatient using a nanoparticle-ligand conjugate. Methods for preparingnanoparticles and their use in delivering pharmaceutical agents havebeen described in U.S. Pat. No. 6,632,671, the disclosures of which areincorporated by reference in their entirety. Methods for preparingnanoparticle-ligand conjugates and their use in deliveringpharmaceutical agents have been described in U.S. Pat. No. 6,372,250,the disclosures of which are incorporated by reference in theirentirety.

The interfering RNA-ligand conjugates and nanoparticle-ligand conjugatescan be administered by intraocular injection, ocular topicalapplication, intravenous injection, oral administration, intramuscularinjection, intraperitoneal injection, transdermal application, ortransmucosal application. The form and concentration in which theconjugate is administered (e.g., capsule, tablet, solution, emulsion)will depend at least in part on the route by which it is administered.

In certain embodiments, the method of treating an ocular diseaseinvolves an ocular disease associated with trabecular meshwork (TM)cells, ciliary epithelium cells, or another cell type of the eye.

In certain embodiments, the invention provides an ocular pharmaceuticalcomposition for preventing or treating an ocular disorder in a patient,comprising an interfering RNA-ligand conjugate or nanoparticle-ligandconjugate of the invention in an ophthalmically acceptable carrier andin a therapeutically effective amount.

Pharmaceutical compositions are formulations that comprise interferingRNAs, or salts thereof, of the invention up to 99% by weight mixed witha physiologically acceptable carrier medium, including those describedinfra, and such as water, buffer, saline, glycine, hyaluronic acid,mannitol, and the like.

Interfering RNA-ligand conjugates and nanoparticle-ligand conjugates ofthe present invention are administered as solutions, suspensions, oremulsions. The following are examples of pharmaceutical compositionformulations that may be used in the methods of the invention.

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Hydroxypropylmethylcellulose 0.5 Sodium chloride 0.8 BenzalkoniumChloride 0.01 EDTA 0.01 NaOH/HCl qs pH 7.4 Purified water (RNase-free)qs 100 mL

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Benzalkonium Chloride 0.01 Polysorbate 800.5 Purified water (RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15(anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05 Cremophor EL 0.1Benzalkonium chloride 0.01 HCl and/or NaOH pH 7.3-7.4 Purified water(RNase-free) q.s. to 100%

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0Phosphate Buffered Saline 1.0 Hydroxypropyl-β-cyclodextrin 4.0 Purifiedwater (RNase-free) q.s. to 100%

As used herein, the term “therapeutically effective amount” refers tothe amount of interfering RNA or a pharmaceutical composition comprisingan interfering RNA determined to produce a therapeutic response in amammal. Such therapeutically effective amounts are readily ascertainedby one of ordinary skill in the art and using methods as describedherein.

Generally, a therapeutically effective amount of the interfering RNAsused in a composition of the invention results in an extracellularconcentration at the surface of the target cell of from 100 pM to 1 μM,or from 1 nM to 100 nM, or from 5 nM to about 50 nM, or to about 25 nM.The dose required to achieve this local concentration will varydepending on a number of factors including the delivery method, the siteof delivery, the number of cell layers between the delivery site and thetarget cell or tissue, whether delivery is local or systemic, etc. Theconcentration at the delivery site may be considerably higher than it isat the surface of the target cell or tissue. Topical compositions can bedelivered to the surface of the target organ, such as the eye, one tofour times per day, or on an extended delivery schedule such as daily,weekly, bi-weekly, monthly, or longer, according to the routinediscretion of a skilled clinician. The pH of the formulation is about pH4.0 to about pH 9.0, or about pH 4.5 to about pH 7.4.

A therapeutically effective amount of a formulation may depend onfactors such as the age, race, and sex of the subject, the severity ofthe disorder, the rate of target gene transcript/protein turnover, theinterfering RNA potency, and the interfering RNA stability, for example.In one embodiment, the interfering RNA is delivered topically to atarget organ and reaches the target mRNA-containing tissue such as thetrabecular meshwork, retina or optic nerve head at a therapeutic dosethereby ameliorating target gene-associated disease process.

Therapeutic treatment of patients with interfering RNAs directed againsttarget mRNAs is expected to be beneficial over small molecule treatmentsby increasing the duration of action, thereby allowing less frequentdosing and greater patient compliance, and by increasing targetspecificity, thereby reducing side effects.

An “ophthalmically acceptable carrier” as used herein refers to thosecarriers that cause at most, little to no ocular irritation, providesuitable preservation if needed, and deliver one or more interferingRNAs of the present invention in a homogenous dosage.

The interfering RNA-ligand conjugates and nanoparticle-ligand conjugatesmay be delivered in solution, in suspension, or in bioerodible ornon-bioerodible delivery devices.

Interfering RNA-ligand conjugates and nanoparticle-ligand conjugates maybe delivered via aerosol, buccal, dermal, intradermal, inhaling,intramuscular, intranasal, intraocular, intrapulmonary, intravenous,intraperitoneal, nasal, ocular, oral, otic, parenteral, patch,subcutaneous, sublingual, topical, or transdermal administration, forexample.

In certain embodiments, treatment of ocular disorders with interferingRNA molecules is accomplished by administration of an interferingRNA-ligand conjugate or nanoparticle-ligand conjugate directly to theeye. Local administration to the eye is advantageous for a number orreasons, including: the dose can be smaller than for systemic delivery,and there is less chance of the molecules silencing the gene target intissues other than in the eye.

A number of studies have shown successful and effective in vivo deliveryof interfering RNA molecules to the eye. For example, Kim et al.demonstrated that subconjunctival injection and systemic delivery ofsiRNAs targeting VEGF pathway genes inhibited angiogenesis in a mouseeye (Kim et al., 2004, Am. J. Pathol. 165:2177-2185). In addition,studies have shown that siRNA delivered to the vitreous cavity candiffuse throughout the eye, and is detectable up to five days afterinjection (Campochiaro, 2006, Gene Therapy 13:559-562).

Interfering RNA-ligand conjugates and nanoparticle-ligand conjugates maybe delivered directly to the eye by ocular tissue injection such asperiocular, conjunctival, subtenon, intracameral, intravitreal,intraocular, anterior or posterior juxtascleral, subretinal,subconjunctival, retrobulbar, or intracanalicular injections; by directapplication to the eye using a catheter or other placement device suchas a retinal pellet, intraocular insert, suppository or an implantcomprising a porous, non-porous, or gelatinous material; by topicalocular drops or ointments; or by a slow release device in the cul-de-sacor implanted adjacent to the sclera (transscleral) or in the sclera(intrascleral) or within the eye. Intracameral injection may be throughthe cornea into the anterior chamber to allow the agent to reach thetrabecular meshwork. Intracanalicular injection may be into the venouscollector channels draining Schlemm's canal or into Schlemm's canal.

For ophthalmic delivery, interfering RNA-ligand conjugates andnanoparticle-ligand conjugates may be combined with ophthalmologicallyacceptable preservatives, co-solvents, surfactants, viscosity enhancers,penetration enhancers, buffers, sodium chloride, or water to form anaqueous, sterile ophthalmic suspension or solution. Solutionformulations may be prepared by dissolving the conjugate in aphysiologically acceptable isotonic aqueous buffer. Further, thesolution may include an acceptable surfactant to assist in dissolvingthe interfering RNA. Viscosity building agents, such as hydroxymethylcellulose, hydroxyethyl cellulose, methylcellulose,polyvinylpyrrolidone, or the like may be added to the compositions ofthe present invention to improve the retention of the compound.

In order to prepare a sterile ophthalmic ointment formulation, theinterfering RNA-ligand conjugate or nanoparticle-ligand conjugate iscombined with a preservative in an appropriate vehicle, such as mineraloil, liquid lanolin, or white petrolatum. Sterile ophthalmic gelformulations may be prepared by suspending the interfering RNA-ligandconjugate or nanoparticle-ligand conjugate in a hydrophilic baseprepared from the combination of, for example, CARBOPOL®-940 (BFGoodrich, Charlotte, N.C.), or the like, according to methods known inthe art. VISCOAT® (Alcon Laboratories, Inc., Fort Worth, Tex.) may beused for intraocular injection, for example. Other compositions of thepresent invention may contain penetration enhancing agents such ascremephor and TWEEN® 80 (polyoxyethylene sorbitan monolaureate, SigmaAldrich, St. Louis, Mo.), in the event the interfering RNA is lesspenetrating in the eye.

In certain embodiments, the invention also provides a kit that includesreagents for attenuating the expression of an mRNA as cited herein in acell. The kit contains an interfering RNA molecule-ligand conjugateand/or the necessary components for interfering RNA molecule-ligandconjugate production (e.g., an interfering RNA molecule as well as theligand and necessary materials for linking). The kit may also containpositive and negative control siRNAs or shRNA expression vectors (e.g.,a non-targeting control siRNA or an siRNA that targets an unrelatedmRNA). The kit also may contain reagents for assessing knockdown of theintended target gene (e.g., primers and probes for quantitative PCR todetect the target mRNA and/or antibodies against the correspondingprotein for western blots). Alternatively, the kit may comprise an siRNAsequence or an shRNA sequence and the instructions and materialsnecessary to generate the siRNA by in vitro transcription or toconstruct an shRNA expression vector.

A pharmaceutical combination in kit form is further provided thatincludes, in packaged combination, a carrier means adapted to receive acontainer means in close confinement therewith and a first containermeans including an interfering RNA composition and a ligand. Such kitscan further include, if desired, one or more of various conventionalpharmaceutical kit components, such as, for example, containers with oneor more pharmaceutically acceptable carriers, additional containers,etc., as will be readily apparent to those skilled in the art. Printedinstructions, either as inserts or as labels, indicating quantities ofthe components to be administered, guidelines for administration, and/orguidelines for mixing the components, can also be included in the kit.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, willappreciate that obvious modifications of the embodiments disclosedherein can be made without departing from the spirit and scope of theinvention. All of the embodiments disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. The full scope of the invention is set out in the disclosureand equivalent embodiments thereof. The specification should not beconstrued to unduly narrow the full scope of protection to which thepresent invention is entitled.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

What is claimed is:
 1. An interfering RNA molecule-ligand conjugate,wherein the ligand is linked to the peptide consisting of SEQ ID NO: 3,and wherein the ligand can bind to a transferrin receptor (TfR).
 2. Theinterfering RNA molecule-ligand conjugate of claim 1, wherein the ligandcomprises a TfR binding domain of transferrin.
 3. The interfering RNAmolecule-ligand conjugate of claim 2, wherein the ligand comprises theTf C-lobe or a TfR-binding portion thereof.
 4. The interfering RNAmolecule-ligand conjugate of claim 1, wherein the interfering RNAmolecule is linked to the ligand via a 7×Arg peptide, 8×Arg peptide,9×Arg peptide, 10×Arg peptide, or 11×Arg peptide.
 5. The interfering RNAmolecule-ligand conjugate of claim 4, wherein the interfering RNAmolecule is linked to the ligand via a 9×Arg peptide.
 6. The interferingRNA molecule-ligand conjugate of claim 1, wherein the interfering RNAmolecule is a siRNA, miRNA, or shRNA.
 7. A pharmaceutical compositioncomprising the interfering RNA molecule-ligand conjugate of claim 1.